RBPs are pivotal regulators of mRNA metabolism, influencing all aspects of post-transcriptional gene control: pre-mRNA splicing, mRNA transport, mRNA stability, and translation. We study if a given RBP associates with a given mRNA by a variety of in vitro binding assays (biotin pulldown, RNA EMSA, surface plasmon resonance/Biacore, etc) and assays to measure binding of endogenous molecules (ribonucleoprotein immunoprecipitation). To investigate RBP function in mammalian cultured cells, we employ approaches such as RBP silencing, RBP overexpression, and the identification of RBP-associated mRNAs using microarrays. We investigate whether RBPs affect the stability of target mRNAs by measuring the steady-state levels and half-lives of the mRNAs of interest as a function of RBP abundance. We investigate whether RBPs affect the translation of target mRNAs by studying the relative assocation of the mRNA with translating polysomes and by quantifying the nascent translation rates of the encoded proteins. We also employ reporter constructs to gain additional insight into the processes modulated by RBPs. During this funding period, we have completed several main projects. In one project, we systematically analyzed the mRNAs that were regulated by AUF1 and discovered that AUF1 interacted with them in pre-mRNA as well as mature mRNA forms (Mazan-Mamczarz et al., 2009). In another, we found evidence that HuR is ubiquitinated in response to heat shock. This modification leads to a loss in HuR abundance (Abdelmohsen et al., 2009). In another project, we found evidence that phosphorylation of HuR at Serine 242 likely contributed to the influence of HuR on the expression of cell cycle regulatory proteins (Kim et al., 2009). Ongoing work is examining the subset of mRNAs that are targets of the RBP NF90, as well as the RBPs that affect differentiation of epithelial cells. We are also continuing with efforts to elucidate the role of HuR in various cancer types.